Clinical impact of rapid molecular detection of respiratory pathogens in patients with acute respiratory infection.

BACKGROUND: Acute respiratory infections (ARI) are a leading cause of morbidity and mortality worldwide. There is a need to demonstrate the clinical impact of using the new, rapid and sensitive molecular assays in prospectively designed studies. OBJECTIVES: To study the impact on medical management of a rapid molecular assay in patients with respiratory infections. STUDY DESIGN: A prospective, randomized, non-blinded study was performed in patients presenting to the Emergency Department during two respiratory seasons (2016-2017). Diagnosis was performed by FilmArray Respiratory Panel (FilmArray-RP) or by immunofluorescence assay (IFA). RESULTS: A total of 432 patients (156 children and 276 adults) were analyzed. Diagnosis with FilmArray-RP was associated with significant changes in medical management including withholding antibiotic prescriptions (OR:15.52, 95%CI:1.99-120.83 in adults and OR:12.23, 95%CI:1.56-96.09 in children), and reduction in complementary studies in children (OR:9.64, 95%CI:2.13-43.63) compared to IFA. Decrease in oseltamivir prescriptions was significantly higher in adults in the FilmArray-RP group (p = 0.042; OR:1.19, 95%CI:0.51-2.79) compared to adults managed with IFA. Diagnostic yield was significantly higher by FilmArray-RP (81%) than by IFA (31%)(p < 0.001). The median time from sample collection to reporting was 1 h 52 min by FilmArray-RP and 26 h by IFA (p < 0.001). CONCLUSIONS: The high respiratory viruses' detection rate and availability of results within two hours when using FilmArray-RP were associated with decreases in antibiotic prescriptions and complementary studies and more accurate use of oseltamivir.

Dynamics of a hepatitis B virus e antigen minus population ascribed to genotype F during the course of a chronic infection despite the presence of anti-HBs antibodies.

The in vivo evolution of genotype F HBV variants was recorded in a chronically infected patient throughout a 3-year observation period. Fluctuating levels of HBs Ag and anti-HBs antibodies were recorded, both of them cocirculating in peripheral blood samples at given times. Fifty S gene derived clones were sequenced and phylogenetically analyzed. As expected, some amino acid replacements within the S ORF were also observed within the P ORF while others were silent for the former. Such change was statistically significant for both S and P overlapping genes, which clearly indicates the appearance of a positive selection pressure. Supporting this notion, amino acid replacements were documented at both B and T cell epitopes in samples from 1997 and 1998. Several mutations were documented within and outside the "a" determinant in the major hydrophilic region. Such substitutions might have resulted from the attempt of HBV to evade both humoral and/or cellular immune response. To the best of our knowledge this unusual profile of HBV variants in presence of usually "neutralizing" anti-HBs antibodies was examined in vivo for the first time.

Cyclic recovery of adenovirus in a stem cell transplant recipient: an inverse association with graft-versus-host disease.

Adenovirus (AdV) infections have been increasingly recognized as significant pathogens that may cause severe morbidity and mortality among stem cell transplant (SCT) recipients. AdV can cause localized infections such as hemorrhagic cystitis (HC), pneumonia, hepatitis and also disseminated disease that can lead to death. We report a case of severe hemorrhagic cystitis in a SCT recipient who died 83 days after transplant. In this patient, AdV recovery was not constantly detected. In fact, fluctuations of the AdV detection in leukocytes and urine were observed by culture and PCR. When analyzing this viral cyclic recovery with different signs or symptoms in the patient, we observed an inverse association with the presence of acute graft-versus-host disease (GVHD). Whether these fluctuations represent donor-derived reactivity, indirectly manifested by the presence of GVHD, requires further study. This is the first case describing a dynamic pattern of AdV replication in leukocytes and urine samples from a patient with severe HC and the temporal correlation with GVHD.

Hepatitis B virus S gene mutants in a patient with chronic active hepatitis with circulating Anti-HBs antibodies.

An adult male farmer with chronic active hepatitis and cirrhosis despite previous circulating anti-HBs antibodies was studied. No markers of other hepatotropic viral infection were observed. HBV DNA was detected in serum by PCR and was characterized further by restriction fragment length polymorphism (RFLP) and sequencing of cloned PCR products derived from the S gene. The HBV DNA was ascribed to genotype F, and single-strand conformational polymorphism (SSCP) demonstrated the co-circulation of multiple quasispecies. Some of the variants exhibited changes located within the neutralizing "a" determinant, located between amino acids 124-147 of the S protein. Within this region, two clones showed either C124R or C124Y mutations. Other mutations were Q129R, C138R, C139R, and S140T (one clone each). Outside the "a" determinant several substitutions were documented. The high degree of the quasispecies variability was probably linked to the severity of the infection. Most members of the patient's family were infected with HBV, all with genotype F.

Multicentered study of viral acute lower respiratory infections in children from four cities of Argentina, 1993-1994.

This study describes the first multicentered study of acute lower respiratory infection viral etiology in young children from four different geographical areas of Argentina. A total of 1,278 children under 5 years of age, hospitalized in primary care centers from Buenos Aires, Córdoba, Santa Fé and Mar del Plata cities during a 2-year period were studied (1993-1994). Nasopharyngeal aspirates were investigated for respiratory syncytial virus (RSV), adenovirus, parainfluenza, and influenza A and B viruses by indirect immunofluorescence. Out of the patients studied, 946 (74%) were under 1 year of age. Viruses were detected in 399 patients (32%). RSV was observed in 25.3% of the samples, representing 78.2% of all viral positive cases. Adenoviruses were detected in 2.5% of the cases, parainfluenza in 2.2%, influenza A in 2.1%, and influenza B in 0.2%. Compared with other viruses, the higher RSV frequency was statistically significant (P < 0.000). Most RSV cases were detected between May and September with a significant peak in July (P < 0.000). Pneumonia was observed in 46% of the patients, bronchiolitis in 41% and other entities in 13%. The case fatality rate observed during the 2 year study was 0.73%. Most of the above respiratory viruses were detected in the four cities, however, the frequency of RSV and influenza were different in the southern city.

[Cytomegalovirus isolation by conventional cell culture and shell vial assay]. / Aislamiento de citomegalovirus por cultivo convencional y cultivo rápido.

In immunocompromised patients, diagnosis of Cytomegalovirus (CMV) active infection is of utmost importance for the initiation, monitoring and ending of antiviral therapy. Therefore, the presence of viral replication should be demonstrated. Isolation in tissue culture is one of the standard methods. The objective of the present paper was to compare two isolation procedures for CMV: conventional cell culture (CC) and rapid shell vial (SV) assay in human fibroblasts. A total of 584 clinical samples were studied between 1991 and 1998. CMV was isolated in 14.4% of the samples, 11.8% of which were positive by SV and 7.7% by CC. Out of 84 positive samples, concordance between both methods was observed in 36% of the cases. We found that 46% of the samples were positive only by SV, while 18% were positive only by CC. The average time required for obtaining the results by CC was 22.6 +/- 2.3 days. Out of the 69 samples positive by SV, 43% were already positive after 24 hours and the rest after 48 hours. These results indicate that SV was more sensitive and rapid than CC. The main advantage of CC, despite its time-consuming process, is the ability to recover the viral strain for both antiviral susceptibility phenotypical tests and strain characterization. Furthermore, in this study, absence of CC would have resulted in the loss of 18% of the positive diagnoses. In conclusion, simultaneous use of both methods is suggested in order to obtain a rapid result and the highest sensitivity.

Aislamiento de citomegalovirus por cultivo convencional y cultivo rápido. / [Cytomegalovirus isolation by conventional cell culture and shell vial assay]

In immunocompromised patients, diagnosis of Cytomegalovirus (CMV) active infection is of utmost importance for the initiation, monitoring and ending of antiviral therapy. Therefore, the presence of viral replication should be demonstrated. Isolation in tissue culture is one of the standard methods. The objective of the present paper was to compare two isolation procedures for CMV: conventional cell culture (CC) and rapid shell vial (SV) assay in human fibroblasts. A total of 584 clinical samples were studied between 1991 and 1998. CMV was isolated in 14.4

of the samples, 11.8

of which were positive by SV and 7.7

by CC. Out of 84 positive samples, concordance between both methods was observed in 36

of the cases. We found that 46

of the samples were positive only by SV, while 18

were positive only by CC. The average time required for obtaining the results by CC was 22.6 +/- 2.3 days. Out of the 69 samples positive by SV, 43

were already positive after 24 hours and the rest after 48 hours. These results indicate that SV was more sensitive and rapid than CC. The main advantage of CC, despite its time-consuming process, is the ability to recover the viral strain for both antiviral susceptibility phenotypical tests and strain characterization. Furthermore, in this study, absence of CC would have resulted in the loss of 18

of the positive diagnoses. In conclusion, simultaneous use of both methods is suggested in order to obtain a rapid result and the highest sensitivity.

Genomic and phylogenetic analysis of hepatitis C virus isolates from argentine patients: a six-year retrospective study.

Typing of hepatitis C virus (HCV) isolates from Argentine patients was performed by using different methodologies in a population of 243 patients. HCV subtype was assigned based upon restriction fragment length polymorphism (RFLP). HCV RNA genomes obtained from serum samples were classified as belonging to clade 1 (53.5%), 2 (23. 0%), or 3 (8.6%); 14.8% of samples showed HCV mixed infections, more frequently implying different subtypes within the same clade. In addition to RFLP typing, phylogenetic relatedness among sequences from both 5' untranslated region (n = 50) and nonstructural 5B coding region (n = 15) was established.

Respiratory syncytial virus: changes in prevalence of subgroups A and B among Argentinian children, 1990-1996.

The frequency of respiratory syncytial virus (RSV) and the distribution of subgroups A and B strains during 7 consecutive years (1990-1996) were examined in two cities of Argentina. Nasopharyngeal aspirates from 1,304 children less than 2 years of age hospitalized with acute lower respiratory infection were studied by indirect immunofluorescence. RSV was detected in 352 cases (26.9%), and the peak activity was observed in midwinter. Subgroup characterization was performed with two monoclonal antibodies against the F protein on nasopharyngeal aspirate smears. Of 195 samples, 174 (89.2%) were identified as subgroup A strains and 21 (10.8%) as subgroup B. Both strains cocirculated during 5 of 7 years studied with subgroup A predominating. Subgroup A occurred at least 8 times as often in all years except for 1994-1995. Children infected by subgroup A were younger than those infected by subgroup B (P < 0.05). The association of subgroup A infection with bronchiolitis and subgroup B with pneumonia was statistically significant (P < 0.03).

Clinical and epidemiologic characteristics of respiratory syncytial virus subgroups A and B infections in Santa Fe, Argentina.

Respiratory Syncytial Virus (RSV) has two major antigenic groups, A and B. The implications of these variants in the epidemiology and pathogenesis of RSV infection are not well defined. This study was undertaken to compare the two RSV subgroups in patients admitted to hospital. Clinical and epidemiologic features of RSV subgroups in children under 30 months of age with proven RSV acute lower respiratory infections were examined during 4 winters from 1993 to 1996 in Santa Fe, Argentina. RSV typing was carried out with monoclonal antibodies in nasopharyngeal cells by indirect immunofluorescence. Of the 177 RSV positive nasopharyngeal aspirates obtained from 1993 to 1996, 85 (48%) were available for typing. Seventy-three (85.9%) specimens were identified as Subgroup A and 12 (14.1%) as Subgroup B. Except in 1993, in which only Subgroup A was detected, both variants circulated throughout the epidemic season. Subgroup A infections produced more severe disease than Subgroup B infections, as assessed by the length of the hospital stay and the use of respiratory support. This difference was age related, being evident in infants 0-6 months old. Patients with Subgroup B infections were also significantly less frequently breast-fed (95% vs. 75% for A and B subgroups, respectively; P = 0.04). It is concluded that the severity of disease in Argentinian patients admitted with acute RSV infections may be associated with Subgroup A strains as determined by a serogrouping method.

Genetic diversity of GBV-C/HGV strains among HIV infected-IVDU and blood donors from Buenos Aires, Argentina.

GBV-C/HGV RNA was investigated in serum samples from 70 HIV(+) intravenous drug users (IVDU), as well as from 200 blood donors from Buenos Aires, Argentina. Viral RNA was demonstrated in 21 IVDU by reverse transcription-nested PCR of the 5' UTR. c-DNA amplified products were analyzed and their sequences compared with those downloaded from GenBank. A phylogenetic tree based on 171 sequences demonstrated the presence of three major genogroups, including two subgroups, within local samples, i.e. group 1 (n=1), 2a (n=11), 2b (n=4) and 3 (n=5). These results agreed entirely with those obtained by a novel RFLP (J. Clin. Microbiol. 37, 1340-1347, 1999) of the same 5' UTR amplicons. As expected, GBV-C/HGV RNA prevalence was significantly higher among IVDU than among blood donors (P<0.0001), although within the latter group an unexpectedly high rate was also detected, since 11 of 200 sera (5.5%) proved positive. These viral isolates were ascribed either to subgroup 2a (n=5), subgroup 2b (n=5) or genogroup 3 (n=1). Briefly, this partial view of GBV-C/HGV molecular epidemiology in Argentina shows: (i) different rates of GBV-C/HGV infection within both IVDU and blood donors; (ii) a high prevalence of viral RNA among blood donors; and (iii) a predominant circulation of genogroup 2, with minor contribution of groups 3 and 1.

Genomic analysis of adenovirus isolated from Argentinian children with acute lower respiratory infections.

BACKGROUND: Adenoviruses are the second cause of acute lower respiratory infection (ALRI) of viral origin in small children from Buenos Aires, Argentina. OBJECTIVE: The aim of this study was to characterize, by restriction enzyme analysis, 17 adenovirus strains isolated from the nasopharyngeal aspirates of children under 2 years of age hospitalized due to ALRI. STUDY DESIGN: Seventeen adenovirus strains isolated between May 1991 and December 1992 in one hospital of Buenos Aires were studied. Adenoviruses were amplified in HEp-2 cells and viral DNA was studied with the restriction enzymes Bam HI and Sma I. RESULTS AND CONCLUSIONS: Eighty two percent (14/17) of the isolates were classified as adenoviruses from subgenus b and 17.7% (3/17) belonged to subgenus c. Genome type 7 h was detected in 85.7% (12/14) and 7 i in 14.3% (2/14) of the strains from subgenus b. The case lethality associated with adenovirus genome type 7 was 28.6% (4/14 cases). Three fatal cases corresponded to Ad 7 h and one to Ad 7i. Ad 7h shows a high prevalence in small children hospitalized with ALRI and is associated with a high fatality rate.

Relevancia clínica de los diferentes métodos diagnósticos para citomegalovirus / Clinical relevance of different idagnostic methods in citomegalovirus diagnosis

This review article details the clinical relevance of different diagnostic methods in citomegalovirus diagnosis

Relevancia clínica de los diferentes métodos diagnósticos para citomegalovirus / Clinical relevance of different idagnostic methods in citomegalovirus diagnosis

This review article details the clinical relevance of different diagnostic methods in citomegalovirus diagnosis

Acute lower respiratory infections due to respiratory syncytial virus and adenovirus among hospitalized children from Argentina.

BACKGROUND: Acute lower respiratory infection (ALRI) is one of the main causes of morbidity and mortality in small children. OBJECTIVE: The aim of this study was to determine the frequency, seasonality and association with clinical entities of respiratory syncytial virus (RSV) and adenoviruses in children with ALRI. STUDY DESIGN: During 2 consecutive years (1991-1992), 168 children under 2 years of age hospitalized due to ALRI in a public pediatric hospital of Buenos Aires, Argentina, were studied. RSV and adenoviruses were investigated on nasopharyngeal aspirates (NPA) by indirect immunofluorescence (IIF). HEp-2 cells were used for adenovirus isolation. RESULTS: RSV was detected in 36.3% and adenoviruses in 14.3% of the cases (P < 0.0001). All adenoviruses detected by IIF were also isolated in culture. Out of 61 RSV cases, 57% corresponded to bronchiolitis and 43% to pneumonia. Ninety-two per cent of children with RSV were less than 1 year old and 70% were less than 5 months. The highest number of RSV cases were observed during winter, with a clear peak in July. Seventy-one per cent of adenovirus cases were associated with pneumonia and only 24% with bronchiolitis (P < 0.02), and predominated in children older than 5 months of age (P < 0.0001). Adenoviruses were detected in almost all months of the year with a small peak at the end of winter and beginning of spring. No significant differences in clinical features at admission, breast feeding or malnutrition were observed among children with RSV or adenovirus diagnosis versus those with no viral etiology. The overall fatality rate was 2.4%. In all fatal cases adenovirus was detected in NPA. Thus, fatality rate among patients with adenoviruses reached 16.7%. CONCLUSIONS: Our findings show the importance of RSV and adenoviruses associated with ALRI in hospitalized children under 2 years of age and the different epidemiological patterns of the two viruses in Buenos Aires, Argentina.

Chlamydia trachomatis en las infecciones respiratorias agudas del tracto inferior en menores de 6 meses / Chlamydia trachomatis in low tract acute respiratory infections in infants under 6 months of age

En este trabajo retrospectivo se presentan los resultados de serología para Chlamydia trachomatis, de detección de sus antígenos así como de infecciones mixtas a virus y clamidia en niños menores de 6 meses con neumonía o bronquiolitis internados en hospitales públicos de Buenos Aires. Se estudiaron 297 sueros pertenecientes a dos poblaciones: a) niños con etiología viral y bacteriana negativa y b) niños en los que no se excluyó previamente etiología viral ni bacteriana. Los anticuerpos anti-C. trachomatis (IgG e IgM) se investigaron por enzimoinmunoensayo (EIE), microinmunofluorescencia (MIF) y/o inmunofluorescencia indirecta (IFI). En 145 aspirados nasofaríngeos (ANF) se investigó Virus Sincicial Respiratorio (RSV) y Adenovirus por IFI. En 25 pacientes con diagnóstico de infección reciente por clamidia se determinó antígeno clamidial en el ANF por EIE. Se determinó la presencia de infección reciente por C. trachomatis en el 15 por ciento de los casos (45/297) mediante la detección de IgM específica, seroconversión para IgG en muestras pareadas y/o títulos elevados y estables de IgG. El 29,6 por ciento de los pacientes presentó IgG (88/297). La frecuencia de infección reciente en las dos poblaciones de niños fue similar. En neumonías se registró una frecuencia significativamente mayor de infección reciente por C. trachomatis (24,3 por ciento) que en bronquiolitis (11,6 por ciento) (p<0,007). En el 40 por ciento de los pacientes con infección reciente por C. trachomatis (10/25) se pudo demostrar la presencia de antígeno clamidial en ANF. El 62 por ciento de los niños con infección reciente fueron menores de 2 meses de edad. En el análisis de las historias clínicas no se observaron diferencias significativas entre los niños con infección reciente por C. trachomatis versus los que presentaron diagnóstico negativo. Se detectó RSV en el 37 por ciento de los casos estudiados, Adenovirus en el 4 por ciento e infección mixta RSV-clamidia en el 5,5 por ciento. Estos resultados señalan la importancia en nuestro medio de C. trachomatis en las neumonías y bronquiolitis en menores de 6 meses y sugiere la necesidad de su diagnóstico rápido para instituir el tratamiento específico.

Chlamydia trachomatis en las infecciones respiratorias agudas del tracto inferior en menores de 6 meses / Chlamydia trachomatis in low tract acute respiratory infections in infants under 6 months of age

En este trabajo retrospectivo se presentan los resultados de serología para Chlamydia trachomatis, de detección de sus antígenos así como de infecciones mixtas a virus y clamidia en niños menores de 6 meses con neumonía o bronquiolitis internados en hospitales públicos de Buenos Aires. Se estudiaron 297 sueros pertenecientes a dos poblaciones: a) niños con etiología viral y bacteriana negativa y b) niños en los que no se excluyó previamente etiología viral ni bacteriana. Los anticuerpos anti-C. trachomatis (IgG e IgM) se investigaron por enzimoinmunoensayo (EIE), microinmunofluorescencia (MIF) y/o inmunofluorescencia indirecta (IFI). En 145 aspirados nasofaríngeos (ANF) se investigó Virus Sincicial Respiratorio (RSV) y Adenovirus por IFI. En 25 pacientes con diagnóstico de infección reciente por clamidia se determinó antígeno clamidial en el ANF por EIE. Se determinó la presencia de infección reciente por C. trachomatis en el 15 por ciento de los casos (45/297) mediante la detección de IgM específica, seroconversión para IgG en muestras pareadas y/o títulos elevados y estables de IgG. El 29,6 por ciento de los pacientes presentó IgG (88/297). La frecuencia de infección reciente en las dos poblaciones de niños fue similar. En neumonías se registró una frecuencia significativamente mayor de infección reciente por C. trachomatis (24,3 por ciento) que en bronquiolitis (11,6 por ciento) (p<0,007). En el 40 por ciento de los pacientes con infección reciente por C. trachomatis (10/25) se pudo demostrar la presencia de antígeno clamidial en ANF. El 62 por ciento de los niños con infección reciente fueron menores de 2 meses de edad. En el análisis de las historias clínicas no se observaron diferencias significativas entre los niños con infección reciente por C. trachomatis versus los que presentaron diagnóstico negativo. Se detectó RSV en el 37 por ciento de los casos estudiados, Adenovirus en el 4 por ciento e infección mixta RSV-clamidia en el 5,5 por ciento. Estos resultados señalan la importancia en nuestro medio de C. trachomatis en las neumonías y bronquiolitis en menores de 6 meses y sugiere la necesidad de su diagnóstico rápido para instituir el tratamiento específico. (AU)

[Chlamydia trachomatis and tubal obstruction sterility]. / Chlamydia trachomatis y esterilidad por obstrucción tubaria.

In order to determine the importance of Chlamydia trachomatis in infertility due to tubal occlusion, the prevalence of C. trachomatis antibody was studied in two populations of Argentine women, pregnant and infertile with tubal occlusion. IgG and IgM against C. trachomatis were determined by indirect immunofluorescence in McCoy cells infected with strain L2Bu434 of C. trachomatis. In infertile women, specific anti-C. trachomatis IgM was detected in 5 out of 32 (15.6%) and IgG in 20 out of 32 (75%). In pregnant women, specific IgM was detected in 4.8% (4/83) and IgG in 20.5% (17/83). The frequency of IgG detection was significantly higher in infertile than in pregnant women (p < 0.000). Thirteen out of 20 (65%) women who underwent laparoscopic examination had evidence of chronic inflammatory disease described as yellowish viscous cysts resembling "frog eggs" in Douglas, utero and/or perihepatitis. Twelve out of fourteen (86%) infertile women with positive serology for C. trachomatis presented one or more of these laparoscopic findings. C. trachomatis antigen was detected in 2 out of 10 tubal biopsies by direct immunofluorescence with monoclonal antibodies although isolation in McCoy cells was negative. Our results show the importance of C. trachomatis in infertility due to tubal occlusion and the need of C. trachomatis diagnosis in routine gynecologic analysis to prevent infertility and neonatal infection.

Chlamydia trachomatis y esterilidad por obstrucción tubaria. / [Chlamydia trachomatis and tubal obstruction sterility]

In order to determine the importance of Chlamydia trachomatis in infertility due to tubal occlusion, the prevalence of C. trachomatis antibody was studied in two populations of Argentine women, pregnant and infertile with tubal occlusion. IgG and IgM against C. trachomatis were determined by indirect immunofluorescence in McCoy cells infected with strain L2Bu434 of C. trachomatis. In infertile women, specific anti-C. trachomatis IgM was detected in 5 out of 32 (15.6

) and IgG in 20 out of 32 (75

). In pregnant women, specific IgM was detected in 4.8

(4/83) and IgG in 20.5

(17/83). The frequency of IgG detection was significantly higher in infertile than in pregnant women (p < 0.000). Thirteen out of 20 (65

) women who underwent laparoscopic examination had evidence of chronic inflammatory disease described as yellowish viscous cysts resembling [quot ]frog eggs[quot ] in Douglas, utero and/or perihepatitis. Twelve out of fourteen (86

) infertile women with positive serology for C. trachomatis presented one or more of these laparoscopic findings. C. trachomatis antigen was detected in 2 out of 10 tubal biopsies by direct immunofluorescence with monoclonal antibodies although isolation in McCoy cells was negative. Our results show the importance of C. trachomatis in infertility due to tubal occlusion and the need of C. trachomatis diagnosis in routine gynecologic analysis to prevent infertility and neonatal infection.